Modified G-protein coupled receptors
AssigneeDuke University
InventorsRobert H. Oakley, Lawrence S. Barak, Stephane A. Laporte, Marc G. Caron
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Abstract
The present invention relates to modified G-protein coupled receptors (GPCRs). The modified GPCRs of the present invention include GPCRs that have been modified to have carboxyl terminal tails comprising one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites. The modified GPCRs of the present invention may comprise a retained portion of a carboxyl-terminus region from a first GPCR fused to a polypeptide, wherein the polypeptide comprises the one or more clusters of phosphorylation. The present invention also relates to methods of screening compounds and sample solutions for GPCR activity using the modified GPCRs.
Claims25
A GPCR comprising an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located downstream of an NPXXY (SEQ ID NO:82) motif of the first GPCR,
- wherein the carboxyl terminal tail portion of different GPCR comprises one or more clusters of phosphorylation sites, and
- wherein the GPCR further comprises a putative site of palmitoylation 10 to 25 amino acid residues downstream of the NPXXY motif (SEQ. ID NO.:
- 82).
A GPCR comprising an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located downstream of an NPXXY (SEQ ID NO:82) motif of the first GPCR,
- wherein the carboxyl terminal tail portion of different GPCR comprises one or more clusters of phosphorylation sites,
- wherein the GPCR further comprises a putative site of palmitoylation 10 to 25 amino acid residues downstream of the NPXXY motif (SEQ. ID NO.:
- 82), and
- wherein the GPCR is conjugated to a detectable molecule.
An isolated nucleic acid encoding a GPCR, wherein the GPCR comprises an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located downstream of an NPXXY (SEQ ID NO:82) motif of the first GPCR,
- wherein the carboxyl terminal tail portion of the different GPCR comprises one or more clusters of phosphorylation sites, and
- wherein the GPCR further comprises a putative site of palmitoylation 10 to 25 amino acid residues downstream of the NPXXY motif (SEQ. ID NO.:
- 82).
An expression vector comprising an isolated nucleic acid encoding a GPCR, wherein the GPCR comprises an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located downstream of an NPXXY (SEQ ID NO:82) motif of the first GPCR,
- wherein the carboxyl terminal tail portion of the different GPCR comprises one or more clusters of phosphorylation sites,
- wherein the GPCR further comprises a putative site of palmitoylation 10 to 25 amino acid residues downstream of the NPXXY motif (SEQ. ID NO.:
- 82), and
- wherein the nucleic acid is operably linked to an expression control sequence.
A host cell comprising an expression vector comprising an isolated nucleic acid encoding a GPCR, wherein the GPCR comprises an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located downstream of an NPXXY (SEQ ID NO:82) motif of the first GPCR,
- wherein the carboxyl terminal tail portion of the different GPCR comprises one or more clusters of phosphorylation sites,
- wherein the GPCR further comprises a putative site of palmitoylation 10 to 25 amino acid residues downstream of the NPXXY motif (SEQ. ID NO.:
- 82), and
- wherein the nucleic acid is operably linked to an expression control sequence.
A GPCR comprising an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located adjacent to a putative site of palmitoylation of the first GPCR,
- wherein the carboxyl terminal tail portion of the different GPCR comprises one or more clusters of phosphorylation sites, and
- wherein the putative site of palmitoylation of the first GPCR is 10 to 25 amino acid residues downstream of an NPXXY motif (SEQ. ID NO.:
- 82) of the first GPCR.
A GPCR comprising an amino acid sequence of a first GPCR modified by substitution of a carboxyl terminal tail portion of the first GPCR with a carboxyl terminal tail portion from a different GPCR, the substitution located adjacent to a putative site of palmitoylation of the first GPCR, the putative site of palmitoylation being a cysteine residue,
- wherein the carboxyl terminal tail portion of the different GPCR comprises one or more clusters of phosphorylation sites, and
- wherein the putative site of palmitoylation of the first GPCR is 10 to 25 amino acid residues downstream of an NPXXY motif (SEQ. ID NO.:
- 82) of the first GPCR.
Dependencies are inferred from the claim text.